To obtain a better understanding of the relationship between differentia- tion and neoplasia our laboratory has been studying the effects of tumor promoting phorbol esters on glial differentiation. Previously we found that phorbol myristate acetate (PMA), the most potent tumor promoter, inhibits the glucocorticoid increase in glycerol phosphate dehydrogenase (GPDH) activity, which is an oligodengroglial specific property in the rat. The effectiveness of phorbol ester analogs to bind to the phorbol ester receptor, protein kinase C, correlates with their effectiveness in inhibiting GPDH induction. In the past year we have found that the natural ligand for the protein kinase C, diacylgylcerols, mimics the phorbol ester effect. Tumor promotion can be divided into two stages. Stage 2 promoters were found to be more active than stage 1 promoters in inhibiting GPDH induction. Binding and degradation studies suggest that the increased activity of stage 1 promoters is either their rate of degradation or the presence of specific stage 2 receptor. Another aspect of glial differentiation being studied is the regulation of intermediary filament expression. During development, astroglial derived cells switch from synthesizing vimentin to synthesizing glial fibrous acidic protein (GFAP). Glioma cells in culture consistently express vimentin and a few cases coexpress vimentin with GFAP. We have not been able to alter the expression of filaments in glioma cells by incubation with differentiating agents. In addition, human glioma cell cultures synthesize vimentin and GFAP when grown as reaggregating cultures, or grown on polylysine or collagen. Primary monolayer cultures of astroglial cells synthesize vimentin and GFAP but synthesize only GFAP when grown as reaggregating cultures. The differences in vimentin synthesis between reaggregating astroglial cell cultures and reaggregating human glioma cultures may reflect changes in regulatory mechanisms which occurred during transformation.